The Na+/H+ exchanger is a ubiquitous transport system with a broad range of cellular functions. These functions depend upon the cell type and the location of the Na+/H+ exchanger within that cell. In the renal proximal tubule, the Na+/H+ exchanger is localized to the apical membrane where it participates in transepithelial Na+ absorption and H+ secretion. In vascular smooth muscle cells, Na+/H+ exchangers participate in pH regulation, vasoconstrictor-induced changes in cell pH, and volume regulation. Because of its many functions, the Na+/H+ exchanger is regulated in many ways. During the first funding period, we studied the Na+/H+ exchanger in renal brush border vesicles and in cultured vascular smooth muscle cells. In the next funding period, we plan to further characterize the mechanisms for regulation of the Na+/H+ exchanger in cultured proximal tubule epithelial cells and vascular smooth muscle cells, with emphasis on protein kinase C-dependent and -independent activation of the Na+/H+ exchanger by vasoactive agents, growth factors, acidosis, and hypokalemia. We hypothesize that activation of the Na+/H+ exchanger by angiotensin II in proximal tubule cells and vascular smooth muscle cells may occur by similar mechanisms or that other features of Na+/H+ exchange in epithelial and mesenchymal cells may be similar. Methods include measurement of cell pH and Ca2+ using fluorescent probes in cultured epithelial and vascular smooth muscle cells, inositol phosphate turnover, arachidonic acid release, and manipulation of G protein function with GTP analogues and specific inhibitors. Studies on the mechanisms of regulation of the Na+/H+ exchanger are important because this transport system is associated with numerous cellular functions like growth, differentiation, vasomotor tone, and cell volume regulation. In addition, alterations in Na+/H+ exchange are associated with pathological states, including hypertension, metabolic acidosis, renal insufficiency, and neoplasia.